Collagen 7 (“C7”) is a structural protein that functions to strengthen and stabilize the skin, and is a major component of anchoring fibrils, which help anchor the top layer of the skin, the epidermis, to the underlying dermis. The C7 monomer assembles as a homotrimer of approximately 900 kDa, containing one NC-1 and one NC-2 binding domains. The homotrimer is held together via an alpha helical coil. From a homotrimer, these C7 protein precursors align into an anti-parallel dimer with an increase in molecular weight to 1.8 mDa. Lateral assembly of anti-parallel dimers leads to the formation of the anchoring fibrils (about 800 nm long).
Epidermolysis bullosa (EB) is a group of genetic conditions that cause the skin to be very fragile and to blister easily. Blisters and skin erosions form in response to minor injury or friction, such as rubbing or scratching. Dystrophic epidermolysis bullosa (DEB) is one of the major forms of epidermolysis bullosa. The signs and symptoms of this condition vary widely among affected individuals. In mild cases, blistering may primarily affect the hands, feet, knees, and elbows. Severe cases of this condition involve widespread blistering that can lead to vision loss, disfigurement, and other serious medical problems. Dystrophic epidermolysis bullosa can be classified into three major types: Recessive dystrophic epidermolysis bullosa (RDEB), non-Hallopeau-Siemens type (non-HS RDEB), and autosomal dominant type (DDEB). Although the types differ in severity, their features overlap significantly and they are all caused by mutations in the COL7A1 gene, which encodes for the protein collagen 7.
The C7 homotrimer can also be purified by the solubilization of amnion using pepsin [2]. The solubilized collagens are partially fractionated by differential salt precipitation, where C7 co-precipitates with collagen 5 (“C5”). Separation of C7 from C5 can be accomplished by CM-cellulose chromatography, following removal of contaminating proteoglycans by DEA-cellulose chromatography. This product is sufficiently pure for routine laboratory use. If further purification is necessary, the pure native structure can be obtained by preparative velocity sedimentation. Pure denatured alpha chains can be obtained following HPLC reversed-phase chromatography.
rC7 has been purified at a laboratory scale by precipitation with ammonium sulfate followed by anion exchange chromatography [2]. However, the yield and purity of this purification method are not sufficient for commercial scale purification of rC7 for therapeutic purposes. Previous attempts to develop a commercial-scale purification method for rC7 had an overall yield of only about 1%.